DE60216944T2 - MICROBICIDE FORMULATION CONTAINING ESSENTIAL OILS OR DERIVATIVES - Google Patents

Abstract

The present invention deals with a microbiocidal aqueous formulation comprising: (i) an effective amount of at least one essential oil component, or derivatives thereof, said derivatives thereof obtained by exposure to light or by oxidation, or mixtures thereof; and (ii) at least one additional stabilizer selected from the group consisting of ethanol in an amount of from 10% to about 50%, an emulsifier, an antioxidant, or an encapsulating agent. The invention is further directed to a method for inhibiting microbial development using said microbiocidal aqueous formulation.

Description

The The present invention relates to agents for inhibiting microbial Growth. In particular, the present invention relates to formulations and methods for inhibiting microbial growth in perishable Agricultural products, household and human hygiene essential oil components or their derivatives, obtained by exposure to light irradiation or by oxidation, together with stabilizers.

The Corruption of perishable agricultural products is caused by microbial Infection caused. Such products are usually over a long enough time periods stored while conditions that increase the number of different species Microorganisms allow and consequently very often a high percentage of the products. In addition to the apparent substantial financial Damage resulting from such corruption will produce some of these Microorganisms toxic and carcinogenic metabolites, which for the people harmful are.

The fight The pathogen infection of perishable agricultural products is becoming nowadays mainly by exogenous application of synthetic fungicides and / or Bactericides achieved. However, these have synthetic chemicals toxic residues in the Products. Furthermore The development of resistant microorganism strains was also noted. As a result, several such fungicides and bactericides by supervisory authorities Gradually withdrawn from circulation. The residual toxicity and the possible Expired for the development of alternatives to synthetic chemicals, currently being used to prevent the spoilage.

Several the alternatives will be described below. For example Irradiation of agricultural products with UV light (S. Ben-Yehoshua, V. Rodov, J.J. Kim and S. Carmeli. Preformed and induced antifungal materials of citrus fruit in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J. Agric. Food Chem. 1992, 40: 1217-1221; V. Rodov, S. Ben-Yehoshua, J.J. Kim, B. Shapiro and Y. Ittah. Ultraviolet illumination induces scoparone production in kumquat and orange fruit and improved decay resistance. J. Amer. Soc. Hortic. Sci. 1992, 117: 188-192) or suspension of the products antagonistic yeasts (C.L. Wilson and E. Chalutz., Postharvest biocontrol of Penicillium rots of citrus with antagonistic yeasts and bacteria. Scientia Horticulturae 1989, 40: 105-112). The UV radiation can However, be phytotoxic and biological control with the antagonistic Yeast is not well accepted commercially, possibly due to insufficient control the pathogens. Furthermore These methods have several disadvantages and some of the most relevant ones health authorities Some of them have not yet been admitted.

Either Citrus fruit as well as various other plants have a certain Endogenous resistance to pathogens, they are the production of antimicrobial Substances in the plant tissues owe (S. Ben-Yehoshua, V. Rodov, J.J. Kim and S. Carmeli. Preformed and induced antifungal materials of citrus fruit in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J. Agric. Food Chem. 1992, 40: 1217-1221; S. Ben-Yehoshua, V. Rodov, D.Q. Fang and J. J. Kim. Preformed antifungal compounds of citrus fruit: effect of postharvest treatments with heat and growth regulators. J. Agric. Food Chem. 1995, 43: 1062-1066; V. Rodov, S. Ben-Yehoshua, D.Q. Fang and J.J. Kim. Preformed antifimgal compounds of lemon fruit: citral and its relation to disease resistance. J. Agric. Food Chem. 1995, 43: 1062-1066). It has already been shown that these substances essential oil components contain a wide range of antimicrobial activity exhibit. U.S. Patents 5,334,619 and 5,958,490 describe the Use of some of course occurring oils as active ingredients to prevent spoilage in agricultural products the harvest. However, only a few of the essential oil components a microbiocidal effect.

Citral [3,7-dimethyl-2,6-octadienal] is an essential oil component, which are found in several types of citrus fruits as well as in some others Plants, such as lemongrass and eucalyptus, are produced naturally. Citral is an unsaturated one Aldehyde from the terpene series and consists of a mixture of isomers of Geranial and Neral. Because of its intense lemon aroma and fragrance Citral in the food and cosmetics industry became vast Scope used. Citral is a safe food additive and is of the U.S. Food and Drug Administration Use in food approved.

It Citral has also been shown to be extremely effective and broad Has a range of antimicrobial and antifungal activity. Actually have Ben-Yehoshua et al. (1992) and Rodov et al. (1995) showed that Citral is the most effective constitutive antifungal compound in lemon fruits.

Limonene, 1-methyl-4- (1-methylethenyl) cyclohexene (also known as p-mentha-1,8-diene), is another example of an abundant essential oil component that can be extracted from citrus oil flavedo oil containers , US 4,379,168 and US 5,951,992 describe the use of limonene as an insecticide or pesticide. In its pure form, however, it has a very low antifungal effect. Chalchat et al. (JC Chalchat, F. Chiron, R. Ph. Garry and Lacoste (J. Essent., Oil Res. 2000, 12, 125-134) disclose the antimicrobial effect of limonene-hydroperoxide on human pathogens.

Aureli et al. (P. Aureli, A. Costantini, and S. Zolea. Antimicrobial activity of some plant essential oil against Listeria monocytogenes. J. Food Protection 1992, 55: 344-348) showed that some essential oil components a strong activity against pathogenic bacteria, such as Listeria, and suggested their use to prevent the infection of foods by Listeria ago.

Several Experiments were carried out to combat citral the corruption of various agricultural products. Citral has been shown to spoil the grains of Aspergillus High moisture barley (B. Nandi, S. Thomke and N. Frieze. Preservation of high moisture barley grains with citral and allyl caproate and preliminary accetability test with piglets. Acta Agric. Scand. 1977, 27: 105-109), raw rice (A.K. Mallick and Nandi. Deterioration of stored rough rice. IV. Preservation and palatability of citral and propionic acid treated grains. Acta Agric. Scand. 1982, 32: 177-187) and Wheat (J. Ghosh and B. Nandi. Preservation of high moisture wheat by some anitfungal volatile compounds and palatability tests with rats. Acta Agric. Scand. 1985, 35: 245-254). Arora and Pandey (R.Arora and G.N. Pandey.The application of essential oils and their isolates for blue mold decay control in citrus reticulata Blanco. J. Food Sci. and tech. 1977, 14: 14-16) reported that Citral, geraniol and other compounds of essential oils the blue sky blight of Citrus reticulata reduce fruit The inventor of the present Ben-Yehoshua, V. Rodov, J.J. Kim, and S. Carmeli. Preformed and induced antifimgal materials of citrus fruits in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J. Agric. Food Chem. 1992, 40: 1217-1221) showed that the application of exogenous citral on lemons inoculated with Penicillium Corruption significantly inhibited.

In most cases the former Use of essential oil components In order to prevent spoilage of agricultural products, the essential oil component in an aqueous Emulsion applied to the products. Although the rotting of the Products partially prevented by the use of such substances became, become essential oil components, including Citral and geraniol, not yet commercially to combat corruption perishable agricultural products. A major reason for not using of these substances is that their application to perishable products, in a concentration effective against the microorganisms, the Harms products, which later Can cause corruption. For example, essential oils cause one impairment the peel in fruits and color changes with meat. This impairment can be serious and leads after a relatively short period of time to considerable corruption of the treated products. Another reason for their lack of commercial Use is their instability, because many of these essential oils are unstable and before the unfolding of their bactericidal action tend to decompose.

The The present invention is based on the fact that essential oil components in food quality or their derivatives, obtained by exposure to light irradiation or by oxidation, as an active ingredient in a stable effective microbicidal formulation for inhibiting microbial growth can be used. In such a formulation, the known phytotoxic damage the essential oil components prevents and stability the essential oil component or their derivatives will be extended, resulting in an environmentally friendly microbicidal composition.

• (i) eine wirksame Menge von mindestens einer essentiellen Ölkomponente, Derivaten davon, wobei die Derivate durch Lichteinwirkung oder durch Oxidation erhalten werden, oder Gemischen davon; und
• (ii) mindestens einen zusätzlichen Stabilisator, ausgewählt aus
• a) einem Emulgator, ausgewählt aus Alkylarylpolyetheralkohol, Polyoxyethylen sorbitanmonolaurat, Polyoxyethylensorbitanmonooleat, Octylphenylpolyetheralkohol oder Gemischen davon,
• b) einem Antioxidans, ausgewählt aus butyliertem Hydroxyanisol (BHA), Ascorbinsäure, butyliertem Hydroxytoluol (BHT), Isoascorbinsäure, α-Tocopherol, β-Karotin oder Gemischen davon, und
• c) einem Mittel zur Verkapselung, ausgewählt aus Maisstärke, Maltodextrin, Silicagel, β-Cyclodextrin, Kasein, Chitosan oder Gemischen davon.

Thus, it is an object of the present invention to provide a novel microbicidal aqueous formulation comprising:

• (i) an effective amount of at least one essential oil component, derivatives thereof, wherein the derivatives are obtained by exposure to light or by oxidation, or mixtures thereof; and
• (ii) at least one additional stabilizer selected from
• a) an emulsifier selected from alkylaryl polyether alcohol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenyl polyether alcohol or mixtures thereof,
• b) an antioxidant selected from butylated hydroxyanisole (BHA), ascorbic acid, butylated hydroxytoluene (BHT), isoascorbic acid, α-tocopherol, β-carotene or mixtures thereof, and
• c) an encapsulating agent selected from corn starch, maltodextrin, silica gel, β-cyclodextrin, casein, chitosan or mixtures thereof.

The essential oil component is from monoterpene hydrocarbons, sesquiterpenes, oxygenated Terpene derivatives, non-terpene derivatives, such as aldehydes, alcohols, acids and phenols, selected. The concentration of essential oils in the aqueous microbicidal composition is about 0.1% to about 1% (v / v). The concentration of the derivative of the essential oil, obtained by exposure to light, is about 1,000 μl / l to about 12,000 μl / l. The microbicidal composition may further comprise an additional Amount of another low-biocide biocide which alone insufficient to inhibit microbial growth is.

• (i) eine wirksame Menge von mindestens einer essentiellen Ölkomponente, Derivaten davon, wobei die Derivate durch Lichteinwirkung oder durch Oxidation erhalten werden, oder Gemischen davon; und (ii) mindestens einen zusätzlichen Stabilisator, ausgewählt aus
• a) einem Emulgator, ausgewählt aus Alkylarylpolyetheralkohol, Polyoxyethylensorbitanmonolaurat, Polyoxyethylensorbitanmonooleat, Octylphenylpolyetheralkohol oder Gemischen davon,
• b) einem Antioxidans, ausgewählt aus butyliertem Hydroxyanisol (BHA), Ascorbinsäure, butyliertem Hydroxytoluol (BHT), Isoascorbinsäure, α-Tocopherol, β-Karotin oder Gemischen davon, und
• c) einem Mittel zur Verkapselung, ausgewählt aus Maisstärke, Maltodextrin, Silicagel, β-Cyclodextrin, Kasein, Chitosan oder Gemischen davon.

It is a further object of the present invention to provide a method for inhibiting microbial growth in perishable agricultural products by applying a microbicidal aqueous formulation comprising:

• (i) an effective amount of at least one essential oil component, derivatives thereof, wherein the derivatives are obtained by exposure to light or by oxidation, or mixtures thereof; and (ii) at least one additional stabilizer selected from
• a) an emulsifier selected from alkylaryl polyether alcohol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenyl polyether alcohol or mixtures thereof,
• b) an antioxidant selected from butylated hydroxyanisole (BHA), ascorbic acid, butylated hydroxytoluene (BHT), isoascorbic acid, α-tocopherol, β-carotene or mixtures thereof, and
• c) an encapsulating agent selected from corn starch, maltodextrin, silica gel, β-cyclodextrin, casein, chitosan or mixtures thereof.

The essential oil component is from monoterpene and sesquiterpene hydrocarbons, oxygenated Terpene derivatives, non-terpene derivatives, such as aldehydes, alcohols, acids and phenols, selected. The Formulation can continue to be another pesticide in a small amount Amount used alone to inhibit microbial growth is insufficient.

A Another object of the present invention is methods of inhibition of microbial growth in the household by application of microbicides aqueous Formulation of the present invention either alone or together usually to provide cleaning agents used.

Yet Another object of the present invention is the use the microbicidal formulation for human hygiene, with the essential oils or derivatives thereof together with suitable additives soap bars, hygiene, dishwashing detergent, mouthwash, Disinfection or cosmetic applications are added.

It is still another object of the present invention, the Use of the microbicidal formulation as nutraceutical for alleviation or treatment of microorganisms caused by light Infections as well as for promotion human health.

Around to understand the invention and to see how they are carried out in practice can now be described a preferred embodiment, only as a non-limiting example, with reference to the attached figures, in which:

1 showing the induction of scoparone production in fully developed green lemon by injecting sun-treated lime, limonene or citral into the albedo or by releasing the contents of the oil container in fully developed green lemons.

2 shows the effect of the fruit ripeness of lemons on the production of phytoalexins in fully developed green and yellow lemon fruits.

3 shows the rate of spoilage of penicillium inoculated grapefruit treated by dipping in formulations of citral and geraniol.

4 the rate of spoilage of Penicillium inoculated lemons treated with an aqueous emulsion of citral stabilized with 25% ethanol, Amif 72 (20% butylated hydroxyanisole, 6% propyl gallate and 4% citric acid in propylene glycol), β-cyclodextrin and polyoxyethylene sorbitan monolaurate ( Tween 20), as compared to the spoilage of fruits of the same species with 5000 ppm Tween 20 without ethanol.

5 the rate of spoilage of penicillium inoculated lemons treated with an aqueous An emulsion of citral comprising octylphenyl polyether alcohol (Triton X 100) and butylated hydroxyanisole (BHA).

6 the rate of spoilage of Penicillium inoculated lemons treated with an aqueous emulsion of citral comprising polyoxyethylene sorbitan monolaurate (Tween 20) and butylated hydroxyanisole (BHA) as compared to the rate of spoilage upon addition of imazalil (Imazalil is a fungicide usually used to protect fruit and vegetables after harvest).

7 the rate of spoilage of penicillium inoculated lemons treated with an aqueous emulsion with 25% ethanol of a crude dichloromethane extract from the flavedo green lemons, compared to 1000 ppm imazalil and 25% ethanol solution.

8th the rate of spoilage of Penicillium inoculated lemons treated with an aqueous solution of 25% ethanol of various combined formulations comprising citral, 1-octanol and dichloromethane crude extract in 25% ethanol from lemon flavedo exposed to sunlight for 4 hours prior to use , shows.

9 the rate of spoilage of Penicillium inoculated lemons treated with a 25% ethanol solution containing 5000 ppm limonene exposed to sunlight for 3 hours. The influence of both duration of dive and number of dives was compared.

10 the rate of spoilage of Penicillium inoculated lemons treated with an aqueous solution of 2500 ppm limonene, which was UV irradiated for three hours prior to its use and subsequently dissolved in 25% ethanol. One minute dipping treatment with this solution (3UVL) was compared with three consecutive 1 minute dips in the same solution with an interval of one hour between these dives (3UVL3). These treatments were compared to dipping in water or in 25% ethanol.

11 shows the effect of limonene hydroperoxide, prepared by photooxidation with Rose Bengal, on the percentage of spoilage of Penicillium digitatum inoculated lemon fruits.

12 shows the effect of limonene hydroperoxide prepared with a molybdate catalyst on the corruption of citrus fruits inoculated with Penicillium digitatum.

13 shows the effect of removing the Rose Bengal catalyst and the dosage of Tween 20 on the phytotoxicity of lemon hydroperoxide-treated citrus fruits.

14 shows the effect of citral formulation on the growth of Staphylococcus aureus cells.

15 shows the effect of citral and sun-treated limonene on the growth of Candida albicans cells.

As stated above, the present invention provides an environmentally friendly microbicidal formulation which is effective in preventing spoilage of agricultural, home, human hygiene and nutraceutical products. The aqueous microbicidal formulation comprises as active ingredient at least one essential oil component or derivatives thereof obtained by exposure to light or by oxidation, or mixtures of such essential oils and / or their derivatives and at least one additional stabilizer selected from an emulsifier, an antioxidant or an agent Encapsulation as mentioned above. The role of the stabilizer is to protect the essential oil components from degradation prior to their microbicidal action and / or to prevent and / or reduce the phytotoxicity of these compounds. The microbicidal essential oil component is selected from monoterpene hydrocarbons and sesquiterpenes, oxygenated terpene derivatives and non-terpene derivatives such as aldehydes (citral or nonanal), alcohols (octanol, nonanol) and phenols (carvacrol). The aqueous microbicidal formulation can be used to effectively control and inhibit microbial growth. All essential oil components are known as food grade components. It should also be emphasized that all components of the aqueous microbicidal formulation are food grade and pose no threat to the human body. A particular far-reaching potential use is for the protection of perishable agricultural products from spoilage caused by microorganisms. Agricultural products may include, for example, all fresh food products that may be spoiled due to microbial infection, such as fruits, vegetables se, meat or fish. Other possible uses for such formulation may be in any case that an effective protection against microorganisms, such as in household use, personal hygiene or nutraceuticals, is needed. For domestic use, the aqueous microbicidal formulation may be used alone or in conjunction with commercial cleaners. For use of the components of the essential oils or their personal hygiene derivatives, an effective amount of this essential oil component or its derivative may be incorporated in soap bars, a cleansing formulation, dishwashing detergent, mouthwash or mouthwash or deodorizing solution or composition. An effective amount of an essential oil component of the present invention, especially for the essential oils selected from citral, perillaaldehyde or limonene; may also be part of a composition used as a nutraceutical. Such nutraceuticals can achieve two effects, protecting against biocidal infections as well as providing additional health benefits, such as the introduction of anti-cancer activity. It has been found that several formulations of the invention comprising essential oils such as citral, limonene, geraniol, menthol, carvone, perrilaaldehyde act as anticancer agents and lower cholesterol and LDL levels. Citral and Citronellal are known to soothe and relax emotions and aid the body in proper digestive function. Such essential oil components are known as phytonutrients or functional foods.

It It is known in the art that some of the essential oil components in the fight against Micro-organisms, in particular agricultural products. However, essential oil components suffer under two inherent Problems that have hitherto limited their practical use. One Problem is with their limited stability, as a result of the oxygenation process, the rapid decomposition of the essential oils in the action of oxygen entails connected. Therefore, although their use as effective Microbicides was actually such a use limited due to their short duration. The use of pretty much huge Quantities of an essential oil, In order to prolong its microbicidal effect, the second disadvantage, which is associated with their use, for Episode: when fruits high concentrations of essential oils, especially when the mixture no real solution is exposed to damage to the products.

It It has now been found that when the essential oil components as the active ingredient of a microbicidal composition together with at least an additive which stabilizes the essential oil components or dissolves, upset to obtain a stable and effective microbicidal composition which is no damage of the products. These additives are made from an antioxidant, a Emulsifier or means for encapsulation selected. Everyone protects these additives the essential oil components through a different mechanism. The emulsifier maintains the formation a microcolloidal solution, the prevention of phytotoxicity of essential oil components supported. The Emulsifiers can alkylaryl polyether alcohol (DX), polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), octylphenyl polyether alcohol (Triton 100) selected become. Some of these emulsifiers are food grade, e.g. B. Tween 20. The concentration of emulsifiers should be above 0.1 % (w / w).

The Antioxidant reduces the oxidation rate leading to its decomposition the essential oils. It also diminishes the inherent phytotoxicity of the essential oil. The antioxidant can be made from compounds such as butylated hydroxyanisole (BHA), ascorbic acid, Isoascorbic acid, α-tocopherol, butylated hydroxytoluene (BHT), β-carotene or mixtures thereof be. Preferred concentrations of the antioxidant in the formulation according to the invention are in the range of 0.05% to 0.8% (w / v). The effect of the antioxidant in reducing the phytotoxicity of the essential oil in case the addition of BHA to citral is shown in Table 1, which the cup defect index it is clear that BHA alone did not cause any deficiency.

The shell defect index is determined by

The defect grade corresponds to 0 = no defects, 1 = slight defects, 2 = medium defects and 3 = severe defects. Table 1: Effect of a 1-minute dip with the antioxidant BHA on shell deficiencies in lemon fruits, stored for 20 days at 20 ° C.

The Formulation added encapsulating agent, together with the essential oil components Complexes which prevent their decomposition and their duration effective mibrobicidal effect is prolonged. The means to Encapsulation can be any kind of food grade matrix or polymer from carbohydrate or protein, or another matrix, like, but not limited on, corn starch, Maltodextrin, β-cyclodextrin, silica gel, Casein, chitosan and their mixtures. Low molecular weight polyethylene and different waxes can also act as materials for encapsulation. Actually became found that adding citral to various wax formulations, that did not contain fungicides, the effectiveness of citral in the Reduction of both spoilage and phytotoxicity increased. Of the Addition of cyclodextrin to citral also had a longer shelf life of citral on the surface the treated orange fruits result. Preferred concentrations of the encapsulating agent in the formulation of the invention are in the range of 0.1 to 0.8% (w / v).

• ¹ Die Früchte wurden 1 Minute in die Emulsion getaucht und anschließend einen Monat bei 20 °C gelagert. Die Phytotoxizität wurde wie in Tabelle 1 gemessen.

The addition of an emulsifier reduces the inherent phytotoxicity of the essential oil. This effect is illustrated by the deficiency index of the essential oil citral with the addition of emulsifier as shown in Table 2. Table 2: Summary of phytotoxicity testing of citral solutions containing emulsifiers (ratio 1: 1 with citral) ¹ .

• ¹ The fruits were immersed in the emulsion for 1 minute and then stored for one month at 20 ° C. The phytotoxicity was measured as in Table 1.

• 1. Verringerung der toxischen Rückstände des Biozids, welche eine entscheidende Anforderung aller Gesundheitsbehörden für alle toxischen Fungizide ist.
• 2. Bekämpfung der Entwicklung einer Resistenz des Pathogens gegen das Biozid.

It should be noted that in addition to the at least one essential oil component, its derivatives obtained by irradiation or mixtures thereof, the microbicidal composition may comprise a further biocide in a very low concentration. Such a low concentration of the biocide alone is not enough to prevent microbial damage, but together with the essential oils of the present invention or their derivatives, microbial spoilage can be prevented. Examples of such biocides are imazalil, thiabendazole, panoctin, rovral, prochloraz, sodium orthophenyl phenate, metalaxyl, al-phosethyl, captan, oxyquinoline, diclorane, benzalkonium chloride, canon, thiophanate methyl, triforine, carbendazim, triadinol, vinclozoline, etaconazole, or mixtures thereof. The concentration of such added pesticides may be from 5 ppm to 100 ppm. The use of such a composition comprising a combination of an essential oil component or its derivatives obtained by exposure to light irradiation together with a small amount of fungicide will provide two important advantages:

• 1. Reduction of toxic residues of biocide, which is a vital requirement of all health authorities for all toxic fungicides.
• 2. Combat the development of resistance of the pathogen to the biocide.

This special point would by using the new formulation with or without the biocide component be achieved. Indeed is the use of another biocide, which is a different mode of action can have, even about a relatively short time, for the recommended way of combating the biocide resistant population.

The essential oil components according to the present invention may be produced synthetically or may be a plant extract preparation comprising a plurality of components of essential oils, ie mixtures thereof. It may also be a purified preparation of natural essential oil enriched with a single essential oil component or a combination thereof. Preparations containing natural essential oils can be made from a variety of plants, such as citrus, lemon grass and eucalyptus trees. Specific non-limiting examples of essential oil compounds include citral, 1-octanol, heptanol, nonanol, geraniol, octanal, nonanal, decanal, perillaaldehyde, perilla alcohol, citronellol, citronellal, carvone, carveol, linalool, vanillin, cinnamaldehyde, cinnamic acid, eugenol, menthol , Limonene, carvacrol, terpineol, thymol, vanillin and camphor. In cases according to the invention wherein the essential oil is derivatized by exposure to light, such derivatization can be made on synthetically produced essential oil, on naturally extracted essential oil or on a crude extract comprising a variety of essential oil. In the latter case, one or more essential oils can be derivatized while others can not be affected. In addition, the Light exposure either before extraction of the essential oil components from their natural source or after their extraction.

According to the present Invention may be a mixture of at least one essential oil component or a derivative obtained therefrom by light irradiation with the Emulsifier and the antioxidant as antimicrobial composition Food, toiletries and household items be added directly to microbicidal purposes. In addition, the mixture in Shape of a liquid or an aerosol by adding non-toxic raw materials in a suitable amount as needed are prepared and added or sprayed for microbicidal purposes.

The Concentration of the at least one essential oil component to achieve an effect needed can easily be determined by the expert in each case be and hang both the nature of the essential oil and the type of Application of the preparation. In the case of citral or geraniol the effective amount is between about 0.1% and 1%, in particular 0.2% and 0.4%.

The Formulation can be applied to the Products at various times before and during their Storage be applied. When the formulation is applied to fruits is preferably before packaging, d. H. after harvest, applied. The formulation of the invention is applied to the products by a process in which the Products containing an effective amount of the formulation, d. H. one Amount of their infection by microorganisms during the inhibited throughout the storage period, are brought into contact. Examples for application methods are diving, gassing, spraying and foaming the fruits in the packaging building. Another method could be Introducing these substances into a wax emulsion. Indeed, as mentioned above, the wax emulsion was a suitable solvent for treatment of different citrus fruits.

One Another application method is fumigating the fruit in a relatively airtight manner Chamber exploiting the volatility many of the active ingredients such as citral.

One other possible Method of application may be by loading the active ingredients Silica gel material used as desiccant or absorbent carried out become. This material could account for more than 10% of its weight absorb our active ingredients and bind as encapsulated substance. However, if the ambient humidity of this material increases, then These effective biocides are released and the water is replaced she. Indeed This is the condition when the fruits in a storage room or in a container for the storage of perishable Agricultural products that continuously transpire included are. Such an application could be slow controlled Release of the fungicide, giving a longer protection against spoilage allows.

The Formulation can also be over slowly degradable polymers are applied, which during their Degrades the essential oil compound contained in them Release products.

According to the present Invention is the pH of the oil formulation preferably acidic, but it can also be basic with a pH up to 9 be.

As mentioned, the aqueous microbicidal formulation should comprise a stabilizer for dissolving the at least one essential oil component. In the case where the stabilizer is ethanol (not claimed), citral at a concentration of 0.2% is found to be much effective in inhibiting the inoculum of non-inoculated fruits of Orange Washington when applied as a formulation with 10-50% ethanol was more effective than when applied in an aqueous emulsion at the same concentration (Table 3, see also Reference Example 4). Table 3: Effects of citral and 50% ethanol on the percentage of spoilage of non-inoculated oranges Washington (numbers =% spoiled fruits)

In addition, occurs no significant impairment of the products usually with the application of essential oil connected is. In the presence of 10 to 50% ethanol caused the essential oil no phytotoxic damage. The injury does not even occur when the essential oils are in relatively high concentrations (0.5 to 1%) known in the art to be cause considerable damage, be applied.

• ¹ 5000 μl/l, 3 Stunden dem Sonnenlicht ausgesetzt in 25%igem Ethanol + 5000 μl/l TWEEN 20

The effect of such formulations in reducing spoilage is thus demonstrated with all the citrus fruit tested in the packaging buildings (results shown in Tables 3 and 4) and with mango fruits and peppers (data not shown). The peppers experiment demonstrated good control of the major pathogens of pepper in Israel, Botrytis cinerea and Alternaria alternata. Table 4: Effect of three hours of sun-exposed limonene on the spoilage of Kumquat fruits after storage for 11 days at 10 ° C and simulation of storage time over 6 days at 20 ° C

• ¹ 5000 μl / l, 3 hours exposed to sunlight in 25% ethanol + 5000 μl / l TWEEN 20

Quantitatively, the essential oil component, limonene, which belongs to the family of monoterpene hydrocarbons, constitutes about 85% of the essential oil components present in the citrus fruit. However, limonene as such is not sufficiently effective. Notwithstanding the fact that it is ineffective, it has been found that limonene can serve as a precursor to a very potent microbicide by exposing homogenized fruit peel to sunlight, followed by extraction of the oil with an organic solvent, especially dichloromethane, hexane or ethyl acetate , The same effect can be seen with UV irradiation of limonene after its extraction ( 9 and 10 ) be achieved. The same applies to synthetically produced limonene. In addition, limonene can also be oxidized using conventional oxidants. A specific non-limiting example is heterogeneous catalysis using molybdate salt.

Therefore may be a microbicidal compound according to the present invention Limonene or a crude extract of essential oils, comprising an essential one Amount of lime irradiated with light include. It should It should be noted that antioxidants when using such derivatives of essential oils, which were exposed to light irradiation should not be used.

Such exposure of limonene to radiation leads to rapid photooxidation, which is high effective antifungal agent. Such a substance may be characterized as the component which gives the purple color in the vanillin-sulfuric acid test (hereinafter "vanillin test") or preferably by blue fluorescence obtained as a result of irradiation with a UV lamp In the case of other terpenes and their derivative compounds, the specific product of limonene can be characterized by its typical color and its retention factor - R _f in the chromatogram - Photooxidation can be effected either by exposing limonene to ultraviolet or white light or sunlight. In these three cases, the resulting material had the same retention time on HPLC These materials gave the same lilac color in the vanillin test and the same spot on a thin layer chromatography plate It could therefore be concluded that all these types of exposure resulted in the same material. It was found that chlorophyll is involved in photooxidation from limonene to the new product, which gives the positive purple color reaction in the vanillin test. This substance showed a great antifungal activity in the bioassay of inhibition of elongation of conidia of Penicillium digitatum. The resulting efficacy was much higher than that of scoparone or scopoletin, known as citrus fruit endogenous phytoalexins, or as that of citral, which was known to be the most effective constitutive fungicide in lemon fruit (Ben-Yehoshua et al., 1992).

Another observation showed that this substance induces the resistance mechanisms of citrus fruits, such as triggering the accumulation of phytoalexins ( 1 ). Treatment of non-inoculated lemons by injecting 5 μl of photo-oxidized limonene into the albedo tissue, just below the flavedo, in these lemons elicited the production of scoparone and scopoletin to levels sufficient to protect the fruits from the pathogen. Similar results were obtained by injecting the dichloromethane or hexane crude extract from lemon flavedo, which was shown to completely prevent spoilage ( 7 ). Similarly, injury to oil containers or injection of limonene into the algae tissue of mature green lemons has had a very important effect in triggering the endogenous resistance mechanisms of citrus fruits, such as the accumulation of scoparone in 1 shown. Such damage to yellow fruits resulted in a much lower response, demonstrating that fruit ripening affects this resistance response and older fruits are less protected ( 2 ). In fact, this lower resistance of yellow fruits was evident in many other experiments. Interestingly, injecting citral did not trigger the same protective reaction ( 1 ). Presumably, unlike lime, citral injection did not induce the production of the vanillin-positive drug. In addition, much higher levels of both scoparone (over 1000 μg / g fresh weight) and scopoletin (over 200 μg / g fresh weight) after dipping inoculated fruit into the crude dichloromethane extract prevented spoilage ( 7 ), detected. The amount of phytoalexin found was several times the amount needed to completely control the pathogens. Thus, the formed limonene hydroperoxides apparently produce reactive oxygen species that activate the plant's immune system. Such reactive oxygen species also affect the pathogen. These reactive oxygen species have a short lifespan and are degraded long before the fruits are put on the market.

One An essential aspect of this new invention is that the spoilage of the Pathogens by both direct inhibition of the pathogen as also by triggering endogenous mechanisms of plant resistance is achieved. Thus, that exercises with Sun or UV treated limonene or sun-treated raw extract direct antifungal effect and also causes the release of the endogenous resistance.

It has previously been reported to have no relevance to the antifungal activity (P. Schieberle, W. Maier, J. Firl and W. Grosch, HRGC separation of hydroperoxides formed during the photosensitized oxidation of (R) - (+) - limonene of High Resolution Chromatography & Chromatography Communications 1987, p 588) that the irradiation of limonene produces various peroxides. It is now shown that the hydroperoxides formed, and in particular (1S, 4R) -p-mentha-2,8-diene-1-hydroperoxide; (1R, 4R) -p-mentha-2,8-dien-1-hydroperoxide; (2R, 4R) -p-mentha-6,8-diene-2-hydroperoxide and (2S, 4R) -p-mentha-6,8-diene-2-hydroperoxide are used as effective biocides in the aqueous solution of the present invention can be. The presence of these hydroperoxides in the irradiated limonene of the present invention was confirmed by gas chromatography / mass spectrometry and nuclear magnetic resonance studies (data not shown). In the present invention, a new process was developed to produce high levels of hydroperoxides with Rose Bengal catalyst or activator that raised the oxygen in the reaction to the singlet oxygen energy level. The result of using this process is that almost all limonene is converted to the hydroperoxides. The mixture of these hydroperoxides was very effective in controlling the spoilage of inoculated lemons even at the dose of 2500 ppm ( 11 ).

The conducted Experiments clearly show that when the crude extract of the shell or if the extracted lime is not irradiated or exposed to sunlight was, the lime solution thus obtained not really enough was effective and too different to inhibit spoilage. Corruption in such cases It developed as if the fruits were not microbicidal at all Formulation would have been brought into contact. Consequently, both the antifungal activity as well as the induction of resistance mechanisms, as shown by the accumulation of scoparone, with these hydroperoxides connected by limonene.

The dichloromethane or hexane extract of lemon flavedo should be exposed to sunlight or irradiated for 3 to 6 hours. The effectiveness of the extract hydroperoxide formulation as a biocidal composition increases when the fruits are exposed to the hydroperoxide solution for a longer time. Dipping the inoculated fruit into a hydroperoxide solution (limonene, exposed to sunlight for 4 hours) for four consecutive 1 minute periods resulted in the prevention of spoilage of inoculated lemons stored at 20 ° C over a period of more than three weeks ( 9 ). The effect of exposing lemons to 3 hours of sunlight and its effectiveness in preventing spoilage compared to no treatment (immersion in water) or treatment with an ethanol solution (25%) are given in Table 4 in a trial that meets commercial conditions , summarized with unvaccinated fruits. In addition, spraying an orchard with 5,000, 10,000 or 20,000 ppm aqueous formulation of limonene in 25% ethanol solution that had been fully converted, reduced the production of scoparone and scopoletin in Valencia fruit on the tree (and after tree removal) the spoilage of these fruits when inoculated with P. digitatum after harvest (data not shown).

In a separate trial showed both citral and sun-treated Limes (solved in 25% ethanol) a marked inhibition of another pathogen, Cladosporium herbarum growing on inoculated corncob.

The conventional Process of corruption control in packaging buildings of citrus fruits several compounds that are also environmentally friendly but technical establish not included in the present formulation. such Compounds are calcium salts, gibberellic acid, 2,4-dichloroacetic acid, acetaldehyde, Chitosan or chitosan plus a low concentration of metals, such as Zn or Cu, some organic acids such as acetic acid, propionic acid, etc. These connections could Be part of our environmentally friendly new formulation. Indeed new data showed such an effect. In fact, the Decrease the pH of the formulations down to 2 to 3 also the fight of the pathogen in experiments with inoculated fruits.

The The invention will now be limited in the following Examples with occasional reference to the accompanying drawings explained in detail.

example 1

Minimal inhibitory Concentration against P. digitatum and mode of action of essential oils in comparison to Imazalil

The minimal inhibitory concentration (MIC) of more than 50 compounds that are in found the citrus oil containers were determined by determining the lowest concentration of Compound in which no growth of the pathogen (in vitro in Petri dishes occurred, evaluated (in vitro). Table 5 gives the MIC the more success promising compounds (compared to imazalil). The necessary Amount of each of the compounds to be tested, dissolved in 0.5 ml of acetone, became Place in a sterile Petri dish (90 mm) containing 15 ml of melted potato dextrose agar (PDA) (50 ° C) to give a final concentration of 1 mg / ml. The shell was gently shaken, for an even distribution to ensure the test compound and the agar was then allowed to solidify. The bowls were under Using a mycelial disc (8 mm) cut from an agar plate culture of P. digitatum, which has not started sporulation yet had, inoculated. The mycelial discs were in the middle of each test dish given and subsequently at 24 ° C calmly. The fungal inhibition was determined by measuring the diameter of the hyphae growth monitored after 7 days.

Tabelle 6: Hemmende Wirkungen verschiedener Verbindungen gegen 3 unterschiedliche Spezies von Zitrusfrüchtenpathogenen im In-vitro-Agardiffusionstest WACHSTUMSHEMMUNGSFLÄCHE DES PATHOGENS¹ (cm²)

• ¹ Die vollständige Hemmung ist die maximale Hemmung, welche 62,3 cm² beträgt.

Another experiment was performed (in vitro to determine if the mode of action was fungicidal or fungistatic) and the mycelial disc of the inoculum from the experimental plate was transferred to a plate containing only PDA and the plates were then rested for the next 5 days Growth controlled. When growth resumed, the compound was classified as fungistatic, and if growth did not recur, it was classified as fungicidal. The results are shown in Tables 5 and 6. Table 5: Minimum inhibitory concentration (MIC) against P. digitatum and modes of action

Table 6: Inhibitory effects of various compounds against 3 different species of citrus fruit pathogens in the in vitro agar diffusion test Growth path of PATHOGEN ¹ (cm ² )

• ¹ Total inhibition is maximum inhibition, which is 62.3 cm ² .

Agar diffusion method

Potato Dextrose Agar (Difco) was prepared and sterilized. The medium was then cooled to 50 ° C in a water bath prior to inoculation by the addition of a suspension of spores of P. digitatum in sterile water to adjust the final concentration of spores in the medium to 10 ⁴ spores / ml. The medium was mixed gently to evenly distribute the spores into each 90 mm diameter petri dish prior to the 15 ml. 5mg of each test substance was pipetted into a 13mm Whatman sterile test paper sterile disc placed centrally on the inoculated agar plate. The plates were incubated at 24 ° C for 3 days. The antifungal effect was controlled by measuring the width of the free zone from the edge of the paper disc to the area of fungal growth. The values represent the radius (mm) of the zone of inhibited growth of the pathogen in the Petri dish. Complete inhibition means that the growth was completely inhibited.

In other tests (data not shown) were found that citral and other formulations of essential oils in the fight the growth of the fungi P. digitatum and the bacteria Erwinia carotovora, a major pathogen of agricultural products.

Reference Example 2

Impeccable grapefruits were harvested from the orchard and re-sampled the same day in the lab to remove damaged fruit. The grapefruits were washed with tap water and air dried. Each fruit was then inoculated by piercing its flavedo tissue 1.5 mm deep with an instrument containing three needles at three different sites. Before each piercing, the instrument was immersed in a spore suspension of Penicillium digitatum (10 ⁶ spores / ml). The inoculated grapefruits were maintained at 17 ° C and 85% relative humidity for 24 hours, after which the grapefruits were divided into six groups and the grapefruits from each group were treated by dipping for 2 minutes in: (a) water; (b) 25% ethanol (EtOH); (c) 0.2% geraniol in 25% EtOH; (d) 0.1% citral in 25% EtOH; (e) 0.2% citral in 25% EtOH and (f) 0.5% citral in 25% EtOH.

The percentage of spoiled fruit in each group was recorded every day after treatment and the results are in 3 shown.

How out 3 As can be seen, the control fruits (submerged only in water) developed rapid spoilage, 100% being reached 8 days after inoculation. The spoilage of fruits dipped in 25% ethanol (EtOH) was delayed, but 6 days after inoculation, a rapid increase of spoilage of EtOH-treated fruit occurred and three weeks after inoculation, more than 50% of the fruit was spoiled.

On the other hand were bearing fruit, dipped in 0.2% geraniol and 0.1-0.2% citral dissolved in 25% EtOH were, a lower rate of spoilage on and on the 18th day after inoculation was spoiled only 20-40% of fruits in these groups. The most effective inhibition of spoilage resulted from dipping of inoculated fruits in 0.2% citral, dissolved in 25% EtOH, which is the corruption of fruits until 18 days after Inoculation completely inhibited and had the consequence that even up 28 days after inoculation showed only 20% of the fruits spoilage. The Dose of 0.5% citral was too high and increased in this trial the corruption due to phytotoxicity.

Example 3

Lemons were inoculated as shown in Reference Example 2 and treated with and without 25% ethanol to test whether the 5000 ppm of Tween 20 emulsifier could stabilize the antifungal activity of citral. The results, presented in 4 , illustrate that this concentration of Tween 20 increased the efficacy of citral. In addition, it prevented the phytotoxicity that is usually caused by citral when it is not dissolved in ethanol. Presumably, the emulsifier allows the formation of a microcolloidal stable emulsion which prevents phytotoxicity by allowing a uniform dispersion of citral, which alone is phytotoxic at these concentrations. Therefore, Tween 20, combined with the antioxidant BHA, could replace the ethanol in the formulation.

Reference Example 4

Not inoculated navel oranges Washington were divided into four groups, which were treated as follows: (a) no treatment; (b) dipped in 50% ethanol; (c) dipped in 0.2% citral in 0.02% emulsifier L-77 (aqueous Emulsion); (d) dipped in 0.2% citral in 50% ethanol and (e) immersed in a 0.2% aqueous Imazalillösung.

The Oranges were then left for four months at 15 ° C in 50-75% relative humidity stored and the percentage of spoiled fruit in each group was recorded at different times after storage. The results are shown in Table 3.

As in Table 3, the percentage was more corrupted fruit after treatment with the formulation (0.2% citral in 50% Ethanol) is substantially lower than that after treatment with either Citral in an aqueous Emulsion or with ethanol alone. Besides, the results are comparable with those obtained with imazalil. The high degree of corruption in the citral treatment with 0.02% Emulsifier was probably caused by phytotoxicity, the result of this treatment due to the lack of either ethanol or a larger amount of emulsifier Was seen.

Example 6

Lemons inoculated with penicillium as shown in Reference Example 2 were immersed for 2 minutes in an aqueous solution comprising citral, emulsifier (Triton X 100) and BHA at various concentrations. The concentration of citral is 1.0%. The control solution comprised 0.5% Triton X 100. The results are in 5 illustrated. The effect of 0.1% BHA in increasing the efficacy of citral to prevent the development of the pathogen is evident.

Example 7

Lemons inoculated with Penicillium as shown in Reference Example 2 were treated with an aqueous solution comprising 0.5% Citral, 0.3% BHA, Tween 20 and Imazalil and stored at 20 ° C for a period of 21 days , The results are in 6 illustrated. Imazalil concentrations of 1 and 10 ppm were insufficient to fully combat spoilage. However, the addition of 0.5% citral to the 10 ppm imazalil completely prevented the spoilage and made it possible to reduce both the dosage and the residues of this effective but undesirable synthetic fungicide. The addition of citral to 1 ppm imazalil did not sufficiently control the spoilage, but was much better than 1 ppm imazalil alone.

Example 8

Lemons inoculated with Penicillium (10 ⁴ spores / ml) were dipped one day later in an aqueous solution comprising citral and a detergent (Tween 20), other essential oils as well as some combinations of the more effective essential oils and stored for a period of 6 days , The results of the microbial protection achieved with these various essential oils are shown in Table 7. Some of these essential oils, especially cinnamic acid, vanillin, octanol and a mixture of several essential oils, were more effective than citral in these experiments. In addition, their effect against P. digitatum persisted longer. Table 7: Effect of various essential oils on the percentage of spoilage of inoculated fruits stored at 20 ° C for 6 days

Example 9

General Method of obtaining one the strongest effective crude extract of lemon flavor

Lemon fruit flavedo (Exokarp) was taken and extracted overnight in dichloromethane, then exposed to sunlight for about 18 hours until the dichloromethane extract turned brown. The flavedo was mixed and filtered through a layer of Whatman paper. The extracted solution was concentrated to remove the dichloromethane and the dense liquid was further separated by chromatography on a column of silica 60 using dichloromethane as carrier. Two dichloromethane fractions, one green and the other colorless, were obtained after chromatography. The active crude extract was isolated by concentration of the green dichloromethane fraction. Lemon fruits inoculated with Penicillium digitatum (10 ⁴ spores / ml) were dipped in an ethanolic solution containing the extracted vanillin-positive compounds 24 hours after inoculation and then stored at 20 ° C. The effect of compounds on pathogen development on the fruits was checked daily during storage. The effect is in 7 illustrates, for various concentrations (10,000, 5,000 and 2,500 ppm crude extract) compared to the effect achieved with an ethanol solution, imazalil or water. The 10,000 ppm crude extract completely prevented the development of the pathogen. In such a high dose of crude extract, some fruits showed some phytotoxicity. However, it was shown that this phytotoxicity was caused mainly by other components and not by the effective antifungal components, since the phytotoxicity could be eliminated, although the biocidal effect was maintained.

Similar Results were obtained with hexane extractions of limonene.

Example 10

Limonene hydroperoxides were over the following two ways made of limonene.

Route 1: Via photooxidation: The conversion of limonene and the yield of the reaction were almost 100% after about 8 hours of reaction time. Pure EtOH was used as solvent, Rose Bengal as Activator. Other light activators, e.g. Chlorophyll, also catalyzes the reaction. A high pressure mercury lamp with a WG 345 filter was used for the photocatalytic reaction. Other ways of light excitation, such as monochromatic light (light emission ≈ 345 nm in the case of Rose Bengal), UV light, laser, also lead to the product limonene hydroperoxide.

Route 2: Via a heterogeneous catalysis path (without light): sodium molybdate (Na ₂ MoO ₄ .2H ₂ O) was used as the catalyst, 300 mL of concentrated H ₂ O ₂ as the oxidant was added to the solvent, to 700 mL of EtOH. added. The reaction conditions to obtain the desired products were: atmospheric pressure, 50 ° C temperature and 5-6 hours reaction time with constant stirring. The conversion of limonene and the yield of the reaction were almost 100%.

The products of the two routes were compared. Gas chromatography measurements showed approximately equal product distributions. The antifungal activity of the products prepared by the two different routes was tested in the same procedure. Lemons inoculated with Penicillium (10 ⁴ spores / ml) were dipped in an aqueous solution comprising limonene hydroperoxide, a detergent (Tween 20) and ethanol. The corruption was assessed at specific intervals over a period of 26 days. The results of the microbial protection achieved with these mixtures are in 11 and 12 shown.

At the Path 1 entered phytotoxicity the fruits after diving on. An increase the amount of Tween 20 in the mixture and removal of Rose Bengal at least significantly curtailed or lessened this phytotoxicity. rose Bengal was replaced by the active hydroperoxide by applying the solution to a Silica column using a mixture of hexane: ethyl acetate 9: 1 separated. After the separation of Rose Bengal was, if at all, a very small phytotoxic damage detected.

The formulation used comprises 0.25% limonene hydroperoxide, 1% Tween 20, 400 ppm Rose Bengal and 25% ethanol. Such a composition completely inhibited spoilage development without any phytotoxicity. At 60 days after inoculation, no spoilage was detected on the treated fruits, whereas in the control sample, the inoculated fruits spoiled after 5 days ( 11 ). This antifungal activity can be demonstrated both with the direct antifungal effect of the limonene hydroperoxide and with the increased amount of scoparone and scopoletin produced by the limonene hydroperoxide as described above for sun-treated limonene ( 1 ).

In route 2, the formulation used comprised 0.5% and 0.25% limonene hydroperoxide, 2% Tween 20 and 25% ethanol. There was no deterioration in inoculated lemons for 12 days ( 12 ). The dose of 0.5% LHPO resulted in complete control of the spoilage and 0.25% LHPO still had some spoilage. The control solution, comprising 2% Tween and 25% ethanol, had very little antifungal activity (90% spoilage), again demonstrating that the active compound is the limonene hydroperoxide.

path 2 is preferred, given that Rose Bengal, the used as catalyst in Route 1 had undesirable effects: he reduces the antifungal effect and increases the phytotoxicity and At the end of the reaction, it must be disconnected from the active compound become.

Example 11

Lemons inoculated with Penicillium (10 ⁴ spores / ml) were dipped in an aqueous solution comprising citral, detergent (Tween 20), other essential oil components, and a sunlight-exposed dichloromethane crude extract of lemon flavedo. The corruption was evaluated at certain intervals over a period of 20 days. The results of the microbial protection achieved with these various essential oils are in 8th shown.

The combination of sun-treated crude extract, citral and 1-octanol in concentrations of 0.5% and 0.25%, respectively, showed inhibition of spoilage development for more than 20 days ( 8th ). These treatments resulted in less than 5% spoilage, while that found in the control solution was greater than 95%. In addition, less than 10% spoilage after treatment occurred with a combination of 0.25% citral and 0.25% 1-octanol or 0.25% citral and 0.25% sun-treated crude extract or 0.125% citral, 0.125% sun-treated Crude extract and 0.125% 1-octanol.

Example 12

Pure lime was exposed to sunlight for three hours. A microbicidal composition was prepared by dissolving the treated limonene in an aqueous solution containing 25% ethanol and a detergent (Tween 20). Lemons inoculated with Penicillium (10 ⁴ spores / ml) were dipped once or up to four times in the composition containing the sun-exposed lime solution one day after inoculation for one minute and stored at 20 ° C. The effect of sun-treated lime on the pathogen development on the fruits was checked daily during storage. 9 shows the effect of the number of dives and the duration of each dive on reducing the spoilage. The data suggest that the results are strongly influenced by the amount of substance absorbed by the pathogen or the fruit tissues. This figure shows that sun-treated lime gives better spoilage control after multiple or longer dives compared to dipping.

Example 13

Pure limonene was irradiated with UV light (254 nm) for three hours. A microbicidal composition was prepared by dissolving the treated limonene in an aqueous solution containing 25% ethanol and a detergent (Tween 20). Lemons inoculated with Penicillium (10 ⁴ spores / ml) were dipped one time after inoculation once or three times in the solution for one minute in the irradiated solution and stored at 20 ° C. In the case of multiple dives, the fruits were allowed to dry for one hour between two consecutive dips.

The effect of the UV irradiation-treated limon on the pathogen development on the fruits was checked during one month. 10 Figure 3 illustrates the similar inhibition of spoilage with the 25% alcohol solution and treatment with 2,500 ppm dipping of UV-treated limonene (designated 3UVL). The three successive dips into the UV-treated limonene (referred to as 3UVL3) showed much better eradication control of all these treatments. The control solution is an aqueous solution containing Tween 20.

Example 14

Wild-type cells Staphylococcus aureus (510 ⁸ , OD ₆₀₀ = 0.219) were cultured for 3 h in broth culture (Cy / Gp) at 37 ° C with different concentrations of extracts of 50-100 μl. The OD was determined at 600 nm. Sample 1 comprises 2000 ppm citral, 25% ethanol, and 2000 ppm Tween 20. Sample 2 comprises the same materials as Sample 1 along with 500 ppm β-CD and 300 ppm BHA. Both samples 1 and 2 significantly inhibited cell growth of Staphylococcus aureus. The same compositions of the substances without citral (Samples 3, 4) did not inhibit the growth of the pathogen ( 14 ).

Example 15

Wild-type cells Candida albicans (1 x 10 ³ ) were grown at 37 ° C on broth culture with different concentrations of extracts of 50-100 μl. The OD was determined at 600 nm.

The following treatments in 6 different tubes were given:
Tube 1: 2000 ppm citral + 25% ethanol + 2000 ppm Tween 20.
Tube 2: 2000 ppm citral + 25% ethanol + 2000 ppm Tween 20 + 300 ppm BHA.
Tube 3: 2000 ppm sun-treated limonene + 2000 ppm Tween 20.
Tube 4: 2000 ppm sun-treated limonene + 25% ethanol + 2000 ppm Tween 20.
Tube 5: 25% ethanol + 2000 ppm Tween 20.
Tube 6: 25% ethanol + 2000 ppm Tween 20 + 500 ppm β-CD + 300 ppm BHA.

2000 ppm citral in 25% ethanol and 2000 ppm Tween 20 or the same materials along with 500 ppm β-CD, 300 ppm BHA or sun-treated limonene dissolved in either 25% ethanol or 2000 ppm Tween 20 completely inhibited cell growth of these organisms. The same compositions of the substances without citral or sun-treated limonene (tubes 5 and 6) did not inhibit the growth of the pathogen ( 15 ).

Claims (10)

Microbicidal aqueous Formulation comprising: (i) an effective amount of at least an essential oil component, Derivatives thereof, wherein the derivatives by the action of light or through Oxidation, or mixtures thereof; and (ii) at least An additional Stabilizer, selected out a) an emulsifier selected from alkylaryl polyether alcohol, Polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, Octylphenyl polyether alcohol or mixtures thereof, b) one Antioxidant, selected from butylated hydroxyanisole (BHA), ascorbic acid, butylated hydroxytoluene (BHT), isoascorbic acid, α-tocopherol, β-carotene or mixtures thereof, and c) a means for encapsulation, selected from cornstarch, Maltodextrin, silica gel, β-cyclodextrin, Casein, chitosan or mixtures thereof. Microbicidal aqueous Formulation according to claim 1, wherein the essential oil component or their derivative selected is from monoterpene or sesquiterpene hydrocarbons, oxygenated terpene derivatives, Non-terpene derivatives such as aldehydes, alcohols, acids, esters and phenols. Microbicidal aqueous Formulation according to claim 2, wherein the essential oil component or their derivative selected is from citral, 1-octanol, heptanol, nonanol, geraniol, octanal, Nonanal, decanal, perillaaldehyde, perilla alcohol, citronellol, citronellal, Carvone, carveol, linalool, vanillin, cinnamaldehyde, cinnamic acid, eugenol, Menthol, limonene, limonene hydroperoxide, carvacrol, terpineol, thymol, Vanillin and camphor or mixtures thereof. Microbicidal aqueous Formulation according to claim 3, wherein the essential oil component or their derivative selected is made of citral, geraniol, limonene and lime hydroperoxide. Microbicidal aqueous Formulation according to claim 4, wherein the concentration of the essential oil component or its derivative 0.1% to 1%. Microbicidal aqueous Formulation according to one the preceding claims, further comprising an additional Biocide at a concentration of about 5 ppm to about 100 ppm. Microbicidal aqueous Formulation according to claim 6, with the biocide selected is imazalil, thiabendazole, panoctin, rovral, prochloraz, sodium orthophenylphenate, Metalaxyl, Al-phosethyl, captan, oxyquinoline, diclorane, benzalkonium chloride, Canon, thiophanatmethyl, triforin, carbendazim, triadimenol, vinclozolin, Etaconazole or mixtures thereof. Microbicidal aqueous Formulation according to claim 7, wherein the essential oil component Citral is the stabilizer is an antioxidant and the extra Biocide is Imazalil. Method for inhibiting microbial growth, comprising the use of an effective amount of an aqueous Formulation according to one the claims 1 to 8. Method according to claim 9 for the protection of fruits and vegetables from corruption after harvest.